Regulation of enzyme function in vivo involves manifold control mechanisms, including those governing protein biosynthesis, transport, activation and catalytic efficiency. The bacterial enzyme alkaline phosphatase is synthesized as an elongated preenzyme, apparently to facilitate transport to the periplasmic space. As isolated, alkaline phosphatase is a dimeric Zn(II) metalloenzyme indicating that the monomeric preenzyme must undergo molecular processing, subunit assembly and metal ion binding to generate the mature enzyme. The sequence of these events and the structural alterations, including changes in the pattern of protein folding accompanying the activation process, is unknown. Induction of alkaline phosphatase synthesis results in appearance of phosphatase and components of the PST phosphate transport system, including a phosphate binding protein, in the periplasmic space. At the molecular level, alkaline phosphatase is subject to allosteric control, as evidenced by the strong negative homotropic interactions which affect substrate and inhibitor binding. The mechanism of action of this enzyme involves the site-specific formation of a phosphoryl-enzyme. The properties of the system indicate that a thermodynamic description of the phosphorylation reaction and the detailed physico-chemical properties of the intermediate an enzyme phosphoserine monoester of unusual structure, can be determined. The objective o the proposed research is to elucidate the nature of these various manifestations of enzymatic regulation with particular emphasis on the manner in which apparently localized events are reflected in structural and energetic alterations of the macromolecule as a whole. The utilization of differential scanning and flow calorimetry, nuclear magnetic resonance methods, X-ray scattering and optical spectroscopy afford unique and complementary advantages in obtaining the data required for a cohesive interpretation. These studies focus on the interrelationships of the preenzyme form of alkaline phosphatase and the mature enzyme and the biochemistry of the functionally related periplasmic phosphate binding protein.